Wearing a traditional black mantilla, she presented her credentials to Pope Francis at the Vatican before officially taking on her role.
The appointment carries some controversy: she is the former speaker's third wife, and had an affair with him when she was a young congressional aide named Callista Bisek and he was still married to his second wife.
In 1999 Gingrich divorced his second wife and the pair married the following year. She is credited with her husband's conversion to Catholicism.
Newt Gingrich was one of the few big-name Republicans to support Donald Trump during the 2016 presidential campaign.
A self-described devout Catholic, Callista Gingrich hopes to work with the Vatican to "defend human rights, advance religious freedom and combat human trafficking," according to a statement released by the US embassy.
On Thursday she and her husband attended the funeral of disgraced American Cardinal Bernard Law at the Vatican.
Law, a once-senior church figure, was forced to resign in 2002 after revelations he failed to stop paedophile priests in one of the biggest crises in American Catholicism. He had been serving in a low-key position in a Rome basilica.
Callista Gingrich, a 51-year-old businesswoman, currently heads Gingrich Productions, a multimedia company and has written a series of children's books.